Exploring sagebrush leaf microbial metagenomes from deep, host-derived sequencing.

BackgroundAs a domesticated species vital to humans, horses are raised worldwide as a source of mechanical energy for sports, leisure, food production, and transportation. The gut microbiota plays an important role in the health, diseases, athletic performance, and behaviour of horses.ResultsHere, using approximately 2.2 Tb of metagenomic sequencing data from gut samples from 242 horses, including 110 samples from the caecum and 132 samples from the rectum (faeces), we assembled 4142 microbial metagenome-assembled genomes (MAG), 4015 (96.93%) of which appear to correspond to new species. From long-read data, we successfully assembled 13 circular whole-chromosome bacterial genomes representing novel species. The MAG contained over 313,568 predicted carbohydrate-active enzymes (CAZy), over 59.77% of which had low similarity match in CAZy public databases. High abundance and diversity of antibiotic resistance genes (ARG) were identified in the MAG, likely showing the wide use of antibiotics in the management of horse. The abundances of at least 36 MAG (e.g. MAG belonging to Lachnospiraceae, Oscillospiraceae, and Ruminococcus) were higher in racehorses than in nonracehorses. These MAG enriched in racehorses contained every gene in a major pathway for producing acetate and butyrate by fibre fermentation, presenting potential for greater amount of short-chain fatty acids available to fuel athletic performance.ConclusionOverall, we assembled 4142 MAG from short- and long-read sequence data in the horse gut. Our dataset represents an exhaustive microbial genome catalogue for the horse gut microbiome and provides a valuable resource for discovery of performance-enhancing microbes and studies of horse gut microbiome.5Q4GzktoVGndspuVBLwUREVideo

The widespread occurrence of sulfate-reducing microorganisms (SRMs) in temporarily oxic/hypoxic aquatic environments indicates an intriguing possibility that SRMs can prevail in constantly oxic/hypoxic terrestrial sulfate-rich environments. However, little attention has been given to this possibility, leading to an incomplete understanding of microorganisms driving the terrestrial part of the global sulfur (S) cycle. In this study, genome-centric metagenomics and metatranscriptomics were employed to explore the diversity, metabolic potential, and gene expression profile of SRMs in a revegetated acidic mine wasteland under constantly oxic/hypoxic conditions. We recovered 16 medium- to high-quality metagenome-assembled genomes (MAGs) containing reductive dsrAB. Among them, 12 and four MAGs belonged to Acidobacteria and Deltaproteobacteria, respectively, harboring three new SRM genera. Comparative genomic analysis based on seven high-quality MAGs (completeness >90% and contamination <10%; including six acidobacterial and one deltaproteobacterial) and genomes of three additional cultured model species showed that Acidobacteria-related SRMs had more genes encoding glycoside hydrolases, oxygen-tolerant hydrogenases, and cytochrome c oxidases than Deltaproteobacteria-related SRMs. The opposite pattern was observed for genes encoding superoxide reductases and thioredoxin peroxidases. Using VirSorter, viral genome sequences were found in five of the 16 MAGs and in all three cultured model species. These prophages encoded enzymes involved in glycoside hydrolysis and antioxidation in their hosts. Moreover, metatranscriptomic analysis revealed that 15 of the 16 SRMs reported here were active in situ. An acidobacterial MAG containing a prophage dominated the SRM transcripts, expressing a large number of genes involved in its response to oxidative stress and competition for organic matter.

Background. The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore exploited culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome, alongside a comparison set from saliva.Results. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae. We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca.Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca.Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria. Although members of the Patescibacteria&#160;are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were, until recently, identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts.Conclusions. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies

The Gemmatimonadota phylum has been widely detected in diverse natural environments, yet their specific ecological roles in many habitats remain poorly investigated. Similarly, the Candidatus ARS69 phylum has been identified only in a few habitats, and literature on their metabolic functions is relatively scarce. In the present study, we investigated the ecological significance of phyla Ca. ARS69 and Gemmatimonadota in the Arctic glacier foreland (GF) ecosystems through genome-resolved metagenomics. We have reconstructed the first high-quality metagenome-assembled genome (MAG) belonging to Ca. ARS69 and 12 other MAGs belonging to phylum Gemmatimonadota from the three different Arctic GF samples. We further elucidated these two groups phylogenetic lineage and their metabolic function through phylogenomic and pangenomic analysis. The analysis showed that all the reconstructed MAGs potentially belonged to novel species. The MAGs belonged to Ca. ARS69 consist about 8296 gene clusters, of which only about 8% of single-copy core genes (n = 980) were shared among them. The study also revealed the potential ecological role of Ca. ARS69 is associated with carbon fixation, denitrification, sulfite oxidation, and reduction biochemical processes in the GF ecosystems. Similarly, the study demonstrates the widespread distribution of different classes of Gemmatimonadota across wide ranges of ecosystems and their metabolic functions, including in the polar region.Key points• Glacier foreland ecosystems act as a natural laboratory to study microbial community structure.• We have reconstructed 13 metagenome-assembled genomes from the soil samples.• All the reconstructed MAGs belonged to novel species with different metabolic processes.• Ca. ARS69 and Gemmatimonadota MAGs were found to participate in carbon fixation and denitrification processes.

Denitrification accounts for a substantial nitrogen loss from environmental systems, shifting microbial composition and impacting other biogeochemical cycles. In Antarctica, rising temperatures cause increased organic matter deposition in marine sediments, which can significantly alter microbially mediated denitrification. To examine the genetic potential of microorganisms driving N-cycling in these sediments, benthic sediment cores were collected at two sites in the Weddell Sea, Antarctica. DNA was extracted from multiple depths at each site, resulting in the reconstruction of 75 high-quality metagenome-assembled genomes (MAGs). Forty-seven of these MAGs contained reductases involved in denitrification. MAGs belonging to the genus Methyloceanibacter were the most abundant MAGs at both sites and all depths, except depth 3-6cmbsf at one site, where they were not identified. The abundance of these Methyloceanibacter MAGs suggests the potential for nitrate-driven methanol oxidation at both sites. MAGs belonging to Beggiatoaceae and Sedimenticolaceae were found to have the genetic potential to produce intermediates in denitrification and the complete pathway for dissimilatory nitrate reduction to ammonia. MAGs within Acidimicrobiia and Dadabacteria had the potential to complete the final denitrification step. Based on MAGs, Antarctic peninsula sediment communities have the potential for complete denitrification and dissimilatory nitrate reduction to ammonia via a consortium.

Little is known regarding the vaginal microbiota of sheep that undergo spontaneous abortions. The aim of this pilot study was to characterize, using 16S rRNA gene sequencing and shotgun metagenomics, the vaginal microbiota throughout the gestation of two ewes (Ewe1 and Ewe2) that spontaneously aborted. To achieve this, weekly vaginal swabs were collected from the ewes prior to breeding until pregnancy testing; thereafter, biweekly swabs were collected until the spontaneous abortion occurred. Based on the 16S rRNA sequencing data, Ewe1’s vaginal microbiota, overall, contained high abundances of Histophilus (12.9% relative abundance), Staphylococcus (10.8% relative abundance) and Unclassified Pasteurellaceae (8.7% relative abundance). Most notable was the high abundance of Campylobacter following the abortion in Ewe1’s vaginal microbiota. Ewe2’s vaginal microbiota was characterized by high abundances of Pasteurella (41.7% relative abundance) throughout gestation. Shotgun metagenomic sequencing produced two high-quality metagenome-assembled genomes (MAGs), identified as Campylobacter jejuni and Histophilus somni. The C. jejuni MAG had 99.95% average nucleotide identity to the most abundant sheep abortive C. jejuni clone in the USA. The H. somni MAG was most similar to a pathogenic H. somni strain and contained genes that contribute to serum resistance and sialic acid utilization. The results presented here demonstrate the need for continued research into the vaginal microbiota, specifically to identify potential predictors of spontaneous abortion.

The leaf microbiome contributes to the functioning of Earth’s ecosystems through interactions with host plants, modifying their health, for better or for worse. The leaf microbiome contains bacteria, fungi, and other microorganisms that interact with each other and are subject to the processes governing community assembly. Microbial community assembly on the leaf is influenced by host plant characteristics, interactions between the different community members, the selective and dispersal-mediated effects of the weather, and time. To study the effects of a leaf microbiome on its host plant it is first important to characterize it; observing the species present and how they are affected by the host plant, weather, and time. For the first chapter of my dissertation, I characterized the fungal leaf microbiome of basin big sagebrush (<italic>Artemisia tridentata</italic> subsp. <italic>tridentata</italic>) leaves over the course on one year, measuring the factors influencing community structure throughout time. I found that the primary factors structuring the sagebrush leaf microbiome are air temperature and the age of the leaf. I discovered that there was a set of fungi living consistently on sagebrush leaves, potentially co-evolving with their host plant over an indeterminate period of time. Additionally, I isolated cultures of microbial species with known effects on the health of other plants, such as the growth promoter <italic>Bacillus amyloliquefaciens</italic>. For my second chapter, I grew sagebrush seedlings in a growth chamber and inoculated them with both a whole natural community of microbes from sagebrush leaves and a single species inoculant of the <italic>B. amyloliquefaciens</italic> isolated from sagebrush in the field. We included two environmental treatments, a tapered drought vs. regular watering, to test the interactive effects of inoculation and stress on sagebrush seedling growth, photosynthesis, and nutrient content. I observed that sagebrush seedlings were susceptible to fungal pathogens during regular watering in a growth chamber, leading to decreased photosynthetic activity and biomass, and that these effects were lessened in the drought conditions. Additionally, <italic>B. amyloliquefaciens</italic> inoculation led to increased percent Nitrogen in the leaves. I noted both positive and negatives effects in our different microbial inoculant groups and that the interactions between microbial inoculation and drought had a positive effect on plant photosynthetic rates. My third chapter was a perspective article. It argues that the potential of leaf microbes to impact entire ecosystems is too great to ignore and should be a consideration when developing management strategies for conservation. I proposed a framework for developing research and application of foliar inoculants in conservation contexts. Together, this dissertation provides the first characterization of the sagebrush leaf microbiome, a highly detailed study of succession of the leaf microbiome over time, a direct observation of environmental context modifying the interaction between a host plant and its leaf microbiome, and a look to the future with the hope that we can include the underappreciated leaf microbiome in conservation strategies. Because interactions (between humans) are also essential to education, I have included a reflection on my educational activities during the course of my Ph.D.

Exercise reduces inflammation, fatigue, and aids overall health. Additionally, physical fitness has been associated with desirable changes in the community composition of the athlete gut microbiome, with health-associated taxa being shown to be increased in active individuals. Here, using a combination of in silico and in vitro methods, we investigate the antimicrobial activity of the athlete gut microbiome. In vitro approaches resulted in the generation of 284 gut isolates with inhibitory activity against Clostridioides difficile and/or Fusobacterium nucleatum, and the most potent isolates were further characterized, and potential bacteriocins were predicted using both MALDI-TOF MS and whole-genome sequencing. Additionally, metagenomic reads from the faecal samples were used to recover 770 Metagenome Assembled Genomes (MAGs), of which 148 were assigned to be high-quality MAGs and screened for the presence of putative bacteriocin gene clusters using BAGEL4 software, with 339 gene clusters of interest being identified. Class I was the most abundant bacteriocin class predicted, accounting for 91.3% of predictions, Class III had a predicted abundance of 7.5%, and Class II was represented by just 1% of all predictions.

Thaumarchaeota constitute an abundant and ubiquitous phylum of Archaea that play critical roles in the global nitrogen and carbon cycles. Most well-characterized members of the phylum are chemolithoautotrophic ammonia-oxidizing archaea (AOA), which comprise up to 5 and 20% of the total single-celled life in soil and marine systems, respectively. Using two high-quality metagenome-assembled genomes (MAGs), here we describe a divergent marine thaumarchaeal clade that is devoid of the ammonia-oxidation machinery and the AOA-specific carbon-fixation pathway. Phylogenomic analyses placed these genomes within the uncultivated and largely understudied marine pSL12-like thaumarchaeal clade. The predominant mode of nutrient acquisition appears to be aerobic heterotrophy, evidenced by the presence of respiratory complexes and various organic carbon degradation pathways. Both genomes encoded several pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases, as well as a form III RuBisCO. Metabolic reconstructions suggest anaplerotic CO2 assimilation mediated by RuBisCO, which may be linked to the central carbon metabolism. We conclude that these genomes represent a hitherto unrecognized evolutionary link between predominantly anaerobic basal thaumarchaeal lineages and mesophilic marine AOA, with important implications for diversification within the phylum Thaumarchaeota.

Anammox is an energy-efficient nitrogen removal process in which anammox bacteria (AnAOB) oxidize NH4+-N to N2 using NO2--N as the electron acceptor. Recent evidence suggests that AnAOB can perform extracellular electron transfer (EET), potentially coupling Fe(III) reduction with NH4+-N oxidation (Feammox). However, whether AnAOB directly participate in Feammox within complex wastewater treatment systems remains unclear. Here, we investigated the iron-mediated nitrogen metabolism pathways in a microaerobic granular sludge (MGS) reactor by integrating enzyme inhibition assays with analyses of gene dynamics and co-occurrence patterns of nitrogen- and iron-cycling genes. Results demonstrate that AnAOB contributed to Feammox activity. The iron reduction gene CT573071, coding a porin-cytochrome c protein complex associated with EET, co-occurred with hao, hzsABC, and hdh genes in Candidatus Kuenenia, suggesting its role in Feammox. Furthermore, four high-quality metagenome-assembled genomes (MAGs) affiliated with Kuenenia stuttgartiensis_A harbored CT573071, hao-like, hzsABC, and hdh genes, along with the hao-cluster, which catalyzes the oxidation of NH4+-N to hydroxylamine. This genomic evidence further supports their dual metabolic capacity. Metatranscriptomic analysis confirmed CT573071 upregulation and its coexpression with the hao, hzsABC, and hdh genes. These findings establish the potential role of K. stuttgartiensis_A in Feammox, providing novel insights into nitrogen removal in low-strength wastewater treatment systems.

Comprehensive protein function annotation is essential for understanding microbiome-related disease mechanisms in the host organisms. However, a large portion of human gut microbial proteins lack functional annotation. Here, we have developed a new metagenome analysis workflow integrating de novo genome reconstruction, taxonomic profiling, and deep learning-based functional annotations from DeepFRI. This is the first approach to apply deep learning-based functional annotations in metagenomics. We validate DeepFRI functional annotations by comparing them to orthology-based annotations from eggNOG on a set of 1,070 infant metagenomes from the DIABIMMUNE cohort. Using this workflow, we generated a sequence catalogue of 1.9 million nonredundant microbial genes. The functional annotations revealed 70% concordance between Gene Ontology annotations predicted by DeepFRI and eggNOG. DeepFRI improved the annotation coverage, with 99% of the gene catalogue obtaining Gene Ontology molecular function annotations, although they are less specific than those from eggNOG. Additionally, we constructed pangenomes in a reference-free manner using high-quality metagenome-assembled genomes (MAGs) and analyzed the associated annotations. eggNOG annotated more genes on well-studied organisms, such as Escherichia coli, while DeepFRI was less sensitive to taxa. Further, we show that DeepFRI provides additional annotations in comparison to the previous DIABIMMUNE studies. This workflow will contribute to novel understanding of the functional signature of the human gut microbiome in health and disease as well as guiding future metagenomics studies. IMPORTANCE The past decade has seen advancement in high-throughput sequencing technologies resulting in rapid accumulation of genomic data from microbial communities. While this growth in sequence data and gene discovery is impressive, the majority of microbial gene functions remain uncharacterized. The coverage of functional information coming from either experimental sources or inferences is low. To solve these challenges, we have developed a new workflow to computationally assemble microbial genomes and annotate the genes using a deep learning-based model DeepFRI. This improved microbial gene annotation coverage to 1.9 million metagenome-assembled genes, representing 99% of the assembled genes, which is a significant improvement compared to 12% Gene Ontology term annotation coverage by commonly used orthology-based approaches. Importantly, the workflow supports pangenome reconstruction in a reference-free manner, allowing us to analyze the functional potential of individual bacterial species. We therefore propose this alternative approach combining deep-learning functional predictions with the commonly used orthology-based annotations as one that could help us uncover novel functions observed in metagenomic microbiome studies.

Wild birds, with their extensive geographic distributions and high mobility, are increasingly recognized as important players in the dissemination of antimicrobial resistance. Their gut microbiota, shaped by exposure to diverse environments, may act as both reservoirs and vectors of antibiotic resistance genes (ARGs), virulence factor genes (VFGs), and mobile genetic elements (MGEs). In this study, we reconstructed 2,516 high-quality metagenome-assembled genomes (MAGs) from 718 gut metagenomes of wild birds to comprehensively profile their resistome and virulome. We identified 5,596 ARG-encoding proteins across 389 distinct ARG types, with multidrug resistance emerging as the most dominant category. Escherichia coli was the principal carrier of ARGs, and genes conferring resistance to elfamycin antibiotics via target alteration were notably widespread—indicating persistent antibiotic selection pressures in avian habitats. Co-occurrence analyses revealed extensive genetic linkage between ARGs, VFGs, and MGEs. Critically, we detected 25 ARG–MGE co-localization events within 5-kilobase genomic regions, highlighting a strong potential for horizontal gene transfer and accelerated resistance dissemination within microbial communities. Of particular concern was the detection of the tetX1 gene—conferring resistance to tigecycline, a last-resort antibiotic—in the gut microbiota of Chroicocephalus ridibundus and Cygnus cygnus. This finding strongly implicates anthropogenic pollution in the spread of clinically relevant ARGs into wildlife and emphasizes the risk of environmental transmission to other hosts, including humans. These results underscore the critical ecological role of wild birds in the global antimicrobial resistance network. As both reservoirs and potential vectors of ARGs, they represent a significant but under-monitored interface between environmental and clinical resistance pathways. Enhanced surveillance and mitigation strategies targeting wildlife are urgently needed to curb the environmental propagation of antimicrobial resistance.

Functional traits and health implications of the global household drinking-water microbiome retrieved using an integrative genome-centric approach

The relative abundance of Acidobacteriia correlated positively with the concentrations of arsenic (As), mercury (Hg), chromium (Cr), copper (Cu) and other metals, suggesting their adaptation of the metal-rich environments. Metagenomic binning reconstructed 29 high-quality metagenome-assembled genomes (MAGs) associated with Acidobacteriia, providing an opportunity to study their metabolic potentials. These MAGs contained genes to transform As, Hg and Cr through oxidation, reduction, efflux and demethylation, suggesting the potential of Acidobacteriia to transform such metal(loid)s. Additionally, genes associated with alleviation of acidic and metal stress were also detected in these MAGs. Acidobacteriia may have the capabilities to resist or transform metal(loid)s in acidic metal-contaminated sites. Moreover, these genes encoding metal transformation could be also identified in the Acidobacteriia-associated MAGs from five additional metal-contaminated sites across Southwest China, as well as Acidobacteriia-associated reference genomes from the NCBI database, suggesting that the capability of metal transformation may be widespread among Acidobacteriia members. This discovery provides an understanding of metabolic potentials of the Acidobacteriia in acidic metal-rich sites.

Societal Impact StatementHeavy metal contamination of soils poses a serious threat to ecosystem health and food security worldwide. This study investigated how native plants, such as Artemisia annua and Buddleja davidii, interact with soil microbes in Pb–Zn waste slag areas. We found that these plants stimulate microbes, which improve phosphorus availability and reduce metal toxicity in the soil. These nature‐based solutions offer a sustainable and eco‐friendly way to restore polluted lands, supporting greener remediation policies and practices for contaminated environments globally.Summary Microorganisms are critical for phosphorus (P) turnover in heavy metal‐contaminated soils, with native plants enhancing these processes by shaping rhizosphere microbial communities. In extreme lead (Pb)–zinc (Zn) slag environments characterized by barrenness and high heavy metal stress, adaptive microbial mechanisms remain underexplored. This study investigates microbial functional genes related to P turnover in the rhizospheres of native plants Carex breviculmis, Artemisia annua, and Buddleja davidii colonizing Pb–Zn waste slag to elucidate the underlying mechanisms. The rhizospheres of A. annua and B. davidii showed increased abundance of genes involved in inorganic P solubilization, indicating their potential for improving soil health. Key P‐cycling genes, including gcd, phnGHKLMN, and phnT, were major predictors of soil available phosphorus (AP), with gcd exhibiting a significant positive correlation with AP. Metagenome‐assembled genomes (MAGs) revealed a diverse community harboring genes for both P cycling and heavy metal resistance. The co‐occurrence of gcd and zntA in Pseudomonadota and Actinomycetota suggests a dual role in P solubilization and metal detoxification, facilitating inorganic P release and efflux of Pb, Zn, and Cd. This gene co‐occurrence may be driven by horizontal gene transfer (HGT), with evidence for 822 potential HGT events detected at the phylum level, including two MAGs acquiring both zntA and gcd. These findings highlight how plant–microbe interactions regulate rhizosphere P cycling in Pb–Zn contaminated soils, demonstrating the potential of A. annua and B. davidii to promote microbial functions that enhance P bioavailability and metal detoxification for ecological remediation.